Subunits of laminin are differentially synthesized in mouse eggs and early embryos

Synthesis of laminin by mouse preimplantation embryos was examined by specific immunoprecipitation of [³⁵S]methionine-labeled cell lysates. The three polypeptide subunits of laminin, A, B₁, and B₂, are not necessarily synchronously expressed during development. Oocytes and eggs synthesize only one immunoprecipitable polypeptide, which comigrates with the intracellular B₁ chains made by PYS cells and contains N-linked oligosaccharide residues which are sensitive to endo-β-N-acetylglucosaminidase H. From the 4- to 8-cell stage, only B₁ and B₂ polypeptides are synthesized, whereas from the 16-cell stage onwards all three laminin polypeptides are made.     

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos

As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. ³H-tryptophan or ³H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of ³H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( ) of the nuclei were densely labelled. Significant accumulation of ³H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae ; gastrulae ). Controls, involving RNase or boiling TCA, demonstrated that the ³H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with ³H-tryptophan or ³H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with ³H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones.In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.     

Isolation and characterization of bone morphogenetic protein-binding proteins from the early Xenopus embryo.

Using a surface plasmon resonance biosensor as a sensitive and specific monitor, we have isolated two distinct bone morphogenetic protein (BMP)-binding proteins, and identified them as lipovitellin 1 and Ep45, respectively. Lipovitellin 1 is an egg yolk protein that is processed from vitellogenin. Both vitellogenin and Ep45 are synthesized under estrogen control in the liver, secreted, and taken up by developing oocytes. In this paper, we have shown that of the TGF-beta family members tested, Ep45 can bind only to BMP-4, whereas lipovitellin 1 can bind to both BMP-4 and activin A. Because of this difference in specificity, we have focused on and further studied Ep45. Kinetic parameters were determined by surface plasmon resonance studies and showed that Ep45 associated rapidly with BMP-4 (k(a) = 1.06 x 10(4) M(-1)s(-1)) and dissociated slowly (k(d) = 1.6 x 10(-4) s(-1)). In Xenopus embryos microinjected with Ep45 mRNA, Ep45 blocked the ability of follistatin to inhibit BMP activity and to induce a secondary body axis in a dose-dependent manner, whereas it had no effect on other BMP antagonists, chordin and noggin. These results support the possibility that Ep45 interacts with BMP to modulate its activities in vivo.     

Regulation of cartilage and bone differentiation by bone morphogenetic proteins.

Quantum advances have recently been made in the understanding of the regulation of cartilage and bone differentiation through the identification, purification, genetic cloning and expression of recombinant bone morphogenetic proteins. Bone morphogenetic proteins are a family of pleiotropic differentiation factors with actions on chemotaxis, mitosis, initiation and promotion of chondrogenic and osteogenic phenotypes. They bind extracellular matrix components, heparin and type IV collagen and initiate bone repair. The cascade of cartilage and bone differentiation consists of several continuous phases: initiation, promotion, maintenance and termination.     

Forkhead proteins are critical for bone morphogenetic protein-2 regulation and anti-tumor activity of resveratrol

Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer.     

A model for cleavage plane determination in early amphibian and fish embryos

Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.     

Delivering on the promise of bone morphogenetic proteins.

The advent of bone growth factors has been widely anticipated since their successful production using recombinant DNA technology. Bone morphogenetic proteins (BMPs) are an important class of bone growth factors and will be the focus of this article. In the near future these therapeutics might revolutionize how clinicians treat such diverse orthopedic applications as the healing of broken bones, increasing bone density lost through aging, and strengthening the spine. These potent proteins require application directly at the site of repair via a delivery system. The choice of delivery system has a profound effect on the clinical outcome. In the past decade, researchers have focused on developing efficient delivery systems and advancing these factors from the bench to the clinic.     

Nuclear variants of bone morphogenetic proteins

Background  

Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts.     

Neurogenesin-1 differentially inhibits the osteoblastic differentiation by bone morphogenetic proteins in C2C12 cells

Bone morphogenetic protein (BMP) antagonists regulate the pleiotropic actions of BMPs by binding to BMPs. We previously isolated the Neurogenesin-1 (Ng1) gene and found that Ng1 protein induces neuronal differentiation in the brain. In this study, we found that Ng1 was expressed in the primordial cells of the skeleton and investigated whether Ng1 protein inhibited the BMP action to induce osteoblastic differentiation in C2C12 myoblasts. Interestingly, Ng1 protein inhibited the BMP7-induced alkaline phosphatase activity while it did not inhibit the BMP2-induced activity. All data suggest that Ng1 protein plays an important role in the embryonic bone formation by differentially regulating BMPs.     

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos

Summary Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.